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General information Notification Number B/IT/24/01 Member State to which the notification was sent Italy Date of acknowledgement from the Member State Competent Authority 18/01/2024 Title of the Project Field trial of NGT1/TEA rice experimental lines for resistance to rice blast Proposed period of release: 30/03/2024 to 26/10/2024 Name of the Institute(s) or Company(ies) Università degli Studi di Milano
Is the same GMPt been notified elsewhere by the same notifier? No Has the same GMPt been notified elsewhere by the same notifier? No Genetically modified plant Complete name of the recipient or parental plant(s):

Common Name	Family Name	Genus	Species	Subspecies	Cultivar/breeding line
Telemaco Ris8imo	Poacee	Oryza	sativa	temperate japonica	Telemaco

Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications: Telemaco Ris8imo plants derive from site-directed mutagenesis using CRISPR/Cas9 technology and the experimental lines arise from independent transformation events (T0) were Cas9 and gRNAs expression was obtained by the insertion of a vector that was then segregated in subsequent generations, maintaining only the deletions produced by the system in the plant's genome. The T0 plants were grown in a confined environment and from them the seeds that will be used for the test were obtained through self-fertilization and segregation of the transgene. The transgene is not present in the plants tested. Telemaco Ris8imo plants have been analyzed through laboratory resistance tests to strains of the fungus Pyricularia oryzae.From a genetic point of view, the variation made consists in the deletion (frameshift) in the genome of portions of the coding sequences of three rice genes. They encode for the proteins Pi21; HMA1 ;HMA2. As a consequence of the genetic deletions, the resulting Telemaco Ris8imo plants present the following new traits (determined in the laboratory): a) ability to produce smaller leaf lesions when infected with strains of the fungus Pyricularia oryzae compared to the same plants without deletions in the Pi21 genes, HMA1 and HMA2; b) no plant growth defects or phenotypic alterations were observed in the laboratory. Any differences in the characteristics of the plants in the field may be highlighted during outdoor experimental tests.Telemaco Ris8imo plants were obtained through genetic transformation mediated by Agrobacterium tumefaciens. Transformed calli resistant to the antibiotic hygromycin were transferred to medium for rooting. The seedlings thus obtained of the T0 generation were transferred to soil for sexual propagation. After genome editing in the T0 generation, the transgene expressing Cas9 and gRNA was segregated and no trace of it was found by PCR in subsequent generations. Telemaco Ris8imo plants are therefore also no longer resistant to hygromicin. The determination of the deletions that occurred at the three expected sites was carried out by PCR analysis and Sanger sequencing. Plants without transgene with deletions at the Pi21 (Os04g0401000), HMA1 (Os04g0469000) and HMA2 (Os04g0464100) sites were selected in homozygosity. The vector used for the genetic transformation expressed the Cas9 protein and 4 specific guide RNAs directed (2 by 2) towards the rice genes Pi21 (2 guide RNAs) and HMA1 and HMA2 (2 guide RNAs). Genetic modification Type of genetic modification: Deletion; In case of deletion of genetic material, give information on the function of the deleted sequences: Telemaco Ris8imo plants present deletions in the coding sequences of Pi21 blast susceptibility gene and HMA1, HMA2 genes. The loss of function of Pi21 (Os04g0401000) is reported in literature for conferring blast resistance. In these plants we also created loss of function mutations in two genes that we named HMA1 and HMA2, that are Os04g0469000 and Os04g0464100 respectively. These are candidates for interacting also with Pyricularia oryzae effectors and therefore also possible susceptibility genes. Brief description of the method used for the genetic modification: Telemaco Ris8imo plants were obtained through genetic transformation mediated by Agrobacterium tumefaciens of rice calli propagated on callus induction medium. These calli were transformed with a vector containing Cas9 coding sequence and two gRNAs plus a selective trait (hygromicin resistance). Calli were then propagated on selection medium supplemented with hygromicin and resistant calli were regenerated. The T0 plants were then moved to rooting medium and then soil. After genome editing in the T0 generation, the transgene expressing Cas9 and gRNA was segregated and no trace of it was found by PCR in subsequent generations. If the recipient or parental plant is a forest tree species, describe ways and extent of dissemination and specific factors affecting dissemination: Not applicable Experimental Release Purpose of the release: Test in an open rice growing field new experimental lines that present deletions in three genes that should confer resistance to rice blast. Blast characteristic lesions will be scored and compared on Telemaco Ris8imo plants and on the parental Telemaco . Geographical location of the site: Società Agricola Federico Radice Fossati, experimental agricultural company, Mezzana Bigli (PV): https://www.google.com/maps/place/Societ%C3%A0+Agricola+Dr+Federico+Radice+Fossati+S.S.+In+ Agriculture/@45.0595408,8.8483011,15z/data=!4m6!3m5!1s0x4787111788797c21:0x15df5c5f19272c0e!8m2!3d45.0595408!4d8.8483011!16s%2Fg%2F1tnj hg8y?entry=ttu Size of the site (m2): 28 m2 Relevant data regarding previous releases carried out with the same GM-plant, if any, specifically related to the potential environmental and human health impacts from the release: Not applicable Environmental Impact and Risk Management Summary of the potential environmental impact from the release of the GMPts: Rice is a cultivated species that requires a very specific agronomic context in order to produce mature seeds. The population of RIS8imo TEA or NGT1 plants at the release site will be fixed by the release technique chosen and cannot increase spontaneously. In addition to using an extremely precautionary buffer or buffer zone in order to prevent the spread of plants subject to occasional crossings, the experimenters will be responsible for removing any other weeds. That said, weeds sexually compatible with Telemaco RIS8imo plants, such as red rice plants, are already naturally resistant to rice blast, so even a possible random crossing with the plants being tested cannot lead to competitive advantages for the weeds.Furthermore, no significant changes are envisaged in the management of agricultural activities and the proposed experimentation will not significantly alter the balance of the soil in the relationship between nitrogen and carbon present. Transfers of the DNA from the three deleted sites to resident soil microorganisms are not conceivable.The purpose of the release is to reduce the use of polluting agrochemicals currently used to combat the rice blast disease. The use of rice NGT1 plants within the RIS8imo project is expected to lead to a decline in the use of these fungicides. The expected interactions are of greater healthiness of the release site and an advantage for the resident microfauna. Brief description of any measures taken for the management of risks: The proposed experimental field is a total of 400 m2 inside which sits the real 28 m2 parcel field where the rice plants from genome editing with CRISPR/Cas9 technology are allocated. The area surrounding the plots with NGT1 plants, the 400 m2 field, is 4 times larger than that theoretically necessary to avoid cross-pollination of statistically significant values with spontaneous plants or sexually compatible weeds, given that rice occasionally has pollination crossed up to 5 meters away. Beyond this limit, the sensitivity limits of the analysis systems fall below.Both sowing and harvesting will be done by hand by staff who have been handling these plants for years. The chosen environment is in the Pavia area, in areas suitable for rice cultivation and potentially afflicted by the rice blast disease.The plants’ residues in the field will be destroyed and buried, the area and agricultural machinery cleaned, the land will not be used for the cultivation of sexually compatible plants for the production of seeds in the 18 months following the experiment. In the event of a natural disaster, the field will be reclaimed and treated with systemic herbicides, the seeds sterilized and the plant residues chopped, incinerated and buried locally. A field notebook will keep track of every process and intervention and appropriate communication will be given to the competent authorities regarding the management and problems of the experimental field. Summary of foreseen field trial studies focused to gain new data on environmental and human health impact from the release: Not applicable Final report - European Commission administrative Information Consent given by the Member State Competent Authority: Yes
03/28/2024